SertePerti Kennel - Comparison of ZHMW and ZMB DNA isolation kits using 40 canine fecal samples with EMU on EMU database
Balázs Kakuk
2025-01-16
Notes
- Unclassified reads were excluded
- Taxa with total reads number of 10 or lower were excluded
- Eukaryotes were excluded from the analysis
- Correlation statistic between ZMB and ZHMW : Pearson’s R^2
- Only the top 25 taxa were used to calculate correlations for each level
- Significantly Different Taxa Identification between ZMB and ZHMW : MAASLIN2
- Statistic to find contribution of factors to the total variance: PERMANOVA
- ALL taxa, NOT just the top 25 were used to calculate the PERMANOVA for each level
- PERMANOVA model: Sex and Date as Fixed Effects and Account for Repeated Measures (Full model)
Import Exp data
Filter Exp data
Filter samples based on total read count
Fix sample names
Add lineage and metadata to Gold Standard Data
Limosilactobacillus fermentum in the theoretical composition was changed to Lactobacillus fermentum
## Error in validObject(.Object) : invalid class "otu_table" object:
## OTU abundance data must have non-zero dimensions.## Error in dimnames(x) <- dn :
## length of 'dimnames' [1] not equal to array extentFix sample names from ‘-’
Merge exp data with GS
Filter Taxa for Gold Standard
These are the taxa that were found in the experimental data
## species taxid superkingdom phylum
## 1 Bacillus subtilis 1423 Bacteria Firmicutes
## 2 Enterococcus faecalis 1351 Bacteria Firmicutes
## 3 Escherichia coli 562 Bacteria Proteobacteria
## 4 Limosilactobacillus fermentum 1613 Bacteria Firmicutes
## 5 Listeria monocytogenes 1639 Bacteria Firmicutes
## 6 Pseudomonas aeruginosa 287 Bacteria Proteobacteria
## 7 Salmonella enterica 28901 Bacteria Proteobacteria
## 8 Staphylococcus aureus 1280 Bacteria Firmicutes
## class order family genus
## 1 Bacilli Bacillales Bacillaceae Bacillus
## 2 Bacilli Lactobacillales Enterococcaceae Enterococcus
## 3 Gammaproteobacteria Enterobacterales Enterobacteriaceae Escherichia
## 4 Bacilli Lactobacillales Lactobacillaceae Limosilactobacillus
## 5 Bacilli Bacillales Listeriaceae Listeria
## 6 Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas
## 7 Gammaproteobacteria Enterobacterales Enterobacteriaceae Salmonella
## 8 Bacilli Bacillales Staphylococcaceae Staphylococcus
## count Gram Stain EXP_sum
## 1 1740000 + NA
## 2 990000 + 73.20904
## 3 1010000 - 6077.60892
## 4 1840000 + NA
## 5 1410000 + NA
## 6 420000 - NA
## 7 1040000 - NA
## 8 1550000 + NAAdd Gram Stain Info
Omit Eukaryotes
Relative abundance of taxa at each taxonomic level
Gram Stain level
Phylum level
Class level
Order level
Family level
Genus level
Species level
Figure XB – no eval
B-diversity Analysis using sample-wise Bray-Curtis distances
Non-metric Multi-Dimensional Scaling (NMDS)
## Square root transformation
## Wisconsin double standardization
## Run 0 stress 0.1470839
## Run 1 stress 0.1646013
## Run 2 stress 0.1718542
## Run 3 stress 0.1825241
## Run 4 stress 0.1601833
## Run 5 stress 0.1721381
## Run 6 stress 0.1722551
## Run 7 stress 0.1841597
## Run 8 stress 0.1590276
## Run 9 stress 0.1616144
## Run 10 stress 0.181801
## Run 11 stress 0.1674317
## Run 12 stress 0.1669914
## Run 13 stress 0.1750451
## Run 14 stress 0.1505306
## Run 15 stress 0.1615029
## Run 16 stress 0.1744525
## Run 17 stress 0.1736455
## Run 18 stress 0.1548971
## Run 19 stress 0.1766343
## Run 20 stress 0.1763632
## *** Best solution was not repeated -- monoMDS stopping criteria:
## 20: stress ratio > sratmaxNMDS v1: color = DNA_isolation_method; shape = Vregion
NMDS v2: color = Vregion; shape = DNA_isolation_method
Statistics: Pearson’s Correlations, PERMANOVA
Parameters and Settings
Correlation
PERMANOVA model
Phylum level
Pearson’s R² between ZHMW or ZMB for each sample
Pairwise comparison of Taxon Abundance R² values between ZHMW and ZMB
## taxon p_value significant label
## <char> <num> <lgcl> <char>
## 1: Actinobacteria 1.117457e-01 FALSE
## 2: Bacteroidetes 1.744864e-07 TRUE *
## 3: Cyanobacteria 3.234749e-01 FALSE
## 4: Deferribacteres 2.941912e-01 FALSE
## 5: Firmicutes 8.161593e-10 TRUE *
## 6: Fusobacteria 2.203607e-07 TRUE *
## 7: Proteobacteria 8.974304e-05 TRUE *
## 8: Synergistetes 3.234749e-01 FALSE
## 9: Tenericutes 1.157477e-01 FALSE
PERMANOVA
Full-model PERMANOVA: source + dna.isolation.method
## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Blocks: strata
## Permutation: free
## Number of permutations: 999
##
## adonis2(formula = otutab_t ~ sex + dna.isolation.methods + date, data = sampdat, method = distance, by = "terms", strata = sampdat$source)
## Df SumOfSqs R2 F Pr(>F)
## sex 1 0.0586 0.00962 1.0000 0.001 ***
## dna.isolation.methods 1 1.1305 0.18547 19.2805 0.001 ***
## date 13 1.1537 0.18927 1.5135 0.051 .
## Residual 64 3.7526 0.61564
## Total 79 6.0954 1.00000
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
Order level
Pearson’s R² between ZHMW or ZMB for each sample
Pairwise comparison of Taxon Abundance R² values between ZHMW and ZMB
## taxon p_value significant label
## <char> <num> <lgcl> <char>
## 1: Acidaminococcales 6.797769e-06 TRUE *
## 2: Aeromonadales 3.016794e-03 TRUE *
## 3: Anaeroplasmatales 1.157477e-01 FALSE
## 4: Bacillales 2.029339e-01 FALSE
## 5: Bacteroidales 1.746399e-07 TRUE *
## 6: Bifidobacteriales 1.673634e-01 FALSE
## 7: Burkholderiales 1.135086e-05 TRUE *
## 8: Campylobacterales 5.593058e-04 TRUE *
## 9: Chroococcidiopsidales 3.234749e-01 FALSE
## 10: Clostridiales 1.765966e-13 TRUE *
## 11: Coriobacteriales 4.849193e-02 TRUE *
## 12: Deferribacterales 2.941912e-01 FALSE
## 13: Eggerthellales 3.811809e-01 FALSE
## 14: Enterobacterales 9.308357e-02 FALSE
## 15: Erysipelotrichales 1.672772e-01 FALSE
## 16: Fusobacteriales 2.203607e-07 TRUE *
## 17: Lactobacillales 9.958588e-03 TRUE *
## 18: Micrococcales 1.914214e-01 FALSE
## 19: Nostocales 3.234749e-01 FALSE
## 20: Oscillatoriales 3.234749e-01 FALSE
## 21: Pleurocapsales 3.234749e-01 FALSE
## 22: Pseudomonadales 2.368003e-01 FALSE
## 23: Selenomonadales 3.791984e-07 TRUE *
## 24: Synechococcales 3.234749e-01 FALSE
## 25: Tissierellales 3.234749e-01 FALSE
## taxon p_value significant label
PERMANOVA
Full-model PERMANOVA: source + dna.isolation.method
## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Blocks: strata
## Permutation: free
## Number of permutations: 999
##
## adonis2(formula = otutab_t ~ sex + dna.isolation.methods + date, data = sampdat, method = distance, by = "terms", strata = sampdat$source)
## Df SumOfSqs R2 F Pr(>F)
## sex 1 0.2456 0.02440 2.8907 0.001 ***
## dna.isolation.methods 1 1.9654 0.19524 23.1302 0.001 ***
## date 13 2.4172 0.24012 2.1882 0.001 ***
## Residual 64 5.4383 0.54023
## Total 79 10.0666 1.00000
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
Genus level
Pearson’s R² between ZHMW or ZMB for each sample
Pairwise comparison of Taxon Abundance R² values between ZHMW and ZMB
## taxon p_value significant label
## <char> <num> <lgcl> <char>
## 1: Acidaminococcales 6.797769e-06 TRUE *
## 2: Aeromonadales 3.016794e-03 TRUE *
## 3: Anaeroplasmatales 1.157477e-01 FALSE
## 4: Bacillales 2.029339e-01 FALSE
## 5: Bacteroidales 1.746399e-07 TRUE *
## 6: Bifidobacteriales 1.673634e-01 FALSE
## 7: Burkholderiales 1.135086e-05 TRUE *
## 8: Campylobacterales 5.593058e-04 TRUE *
## 9: Chroococcidiopsidales 3.234749e-01 FALSE
## 10: Clostridiales 1.765966e-13 TRUE *
## 11: Coriobacteriales 4.849193e-02 TRUE *
## 12: Deferribacterales 2.941912e-01 FALSE
## 13: Eggerthellales 3.811809e-01 FALSE
## 14: Enterobacterales 9.308357e-02 FALSE
## 15: Erysipelotrichales 1.672772e-01 FALSE
## 16: Fusobacteriales 2.203607e-07 TRUE *
## 17: Lactobacillales 9.958588e-03 TRUE *
## 18: Micrococcales 1.914214e-01 FALSE
## 19: Nostocales 3.234749e-01 FALSE
## 20: Oscillatoriales 3.234749e-01 FALSE
## 21: Pleurocapsales 3.234749e-01 FALSE
## 22: Pseudomonadales 2.368003e-01 FALSE
## 23: Selenomonadales 3.791984e-07 TRUE *
## 24: Synechococcales 3.234749e-01 FALSE
## 25: Tissierellales 3.234749e-01 FALSE
## taxon p_value significant label
PERMANOVA
Full-model PERMANOVA: source + dna.isolation.method
## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Blocks: strata
## Permutation: free
## Number of permutations: 999
##
## adonis2(formula = otutab_t ~ sex + dna.isolation.methods + date, data = sampdat, method = distance, by = "terms", strata = sampdat$source)
## Df SumOfSqs R2 F Pr(>F)
## sex 1 0.3503 0.02341 2.8762 0.001 ***
## dna.isolation.methods 1 3.2119 0.21461 26.3718 0.001 ***
## date 13 3.6093 0.24116 2.2796 0.001 ***
## Residual 64 7.7948 0.52082
## Total 79 14.9663 1.00000
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
Species level
Pearson’s R² between ZHMW or ZMB for each sample
Pairwise comparison of Taxon Abundance R² values between ZHMW and ZMB
## taxon p_value significant label
## <char> <num> <lgcl> <char>
## 1: Anaerobiospirillum succiniciproducens 4.820577e-03 TRUE *
## 2: Blautia glucerasea 1.705144e-11 TRUE *
## 3: Blautia hansenii 3.010974e-01 FALSE
## 4: Blautia producta 1.055413e-08 TRUE *
## 5: Blautia sp. N6H1-15 1.220307e-04 TRUE *
## 6: Clostridium perfringens 5.166682e-01 FALSE
## 7: Enterocloster bolteae 1.313457e-09 TRUE *
## 8: Faecalibacterium prausnitzii 7.687366e-06 TRUE *
## 9: Faecalimonas umbilicata 1.039283e-07 TRUE *
## 10: Fusobacterium perfoetens 3.881664e-07 TRUE *
## 11: Fusobacterium varium 2.208536e-06 TRUE *
## 12: Lactobacillus animalis 1.562315e-02 TRUE *
## 13: Lactobacillus johnsonii 5.637611e-01 FALSE
## 14: Lactobacillus reuteri 1.498909e-01 FALSE
## 15: Megamonas funiformis 7.241348e-05 TRUE *
## 16: Megamonas hypermegale 2.581652e-08 TRUE *
## 17: Megamonas rupellensis 7.436912e-07 TRUE *
## 18: Peptacetobacter hiranonis 6.127696e-13 TRUE *
## 19: Phascolarctobacterium succinatutens 6.797769e-06 TRUE *
## 20: Phocaeicola plebeius 1.918687e-07 TRUE *
## 21: Romboutsia timonensis 7.379722e-01 FALSE
## 22: Streptococcus equinus 6.336182e-02 FALSE
## 23: Streptococcus lutetiensis 4.101400e-02 TRUE *
## 24: Turicibacter sp. H121 1.922370e-01 FALSE
## 25: [Ruminococcus] gnavus 9.990586e-01 FALSE
## taxon p_value significant label
PERMANOVA
Full-model PERMANOVA: source + dna.isolation.method
## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Blocks: strata
## Permutation: free
## Number of permutations: 999
##
## adonis2(formula = otutab_t ~ sex + dna.isolation.methods + date, data = sampdat, method = distance, by = "terms", strata = sampdat$source)
## Df SumOfSqs R2 F Pr(>F)
## sex 1 0.3730 0.02338 2.7969 0.001 ***
## dna.isolation.methods 1 3.2992 0.20679 24.7391 0.001 ***
## date 13 3.7468 0.23485 2.1612 0.001 ***
## Residual 64 8.5350 0.53498
## Total 79 15.9539 1.00000
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
Summarised R² values Across Taxon Levels
Barplot
Lineplot
Summary of PERMANOVAs on Different Rank Levels
